Treating glaucoma, cardiovascular diseases, and renal diseases

ABSTRACT

This document provides methods and materials related to treating glaucoma, ocular hypertension, cardiovascular diseases, and renal diseases. For example, this document provides isolated nucleic acid molecules and viral vectors (e.g., lentiviral vectors) containing isolated nucleic acid molecules. Methods for reducing intraocular pressure as well as symptoms and progression of cardiovascular and renal diseases also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/624,383, filedSep. 21, 2012, which is a continuation application of U.S. Ser. No.12/298,431, filed Aug. 6, 2009 (now U.S. Pat. No. 8,299,043), which is aNational Stage application under 35 U.S.C. §371 and claims benefit ofInternational Application No. PCT/US2007/067710 having an InternationalFiling Date of Apr. 27, 2007, which claims the benefit of priority ofU.S. Provisional Application No. 60/795,789 having a filing date of Apr.28, 2006. The disclosures of the prior applications are considered partof (and are incorporated by reference in) the disclosure of thisapplication.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under EY014411 awardedby the National Institutes of Health. The government has certain rightsin the invention,

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treatingglaucoma, cardiovascular diseases, and renal diseases. For example, thisdocument relates to methods and materials that can be used to reduceintraocular pressure.

2. Background Information

Glaucoma is characterized by a loss of visual function due to damage tothe optic nerve. The several morphologically or functionally distincttypes of glaucoma are typically characterized by elevated intraocularpressure, which is considered to be causally related to the pathologicalcourse of the disease. Ocular hypertension is a condition whereintraocular pressure is elevated but no apparent loss of visual functionhas occurred. Such patients are considered to be at high risk for theeventual development of the visual loss associated with glaucoma. Ifglaucoma or ocular hypertension is detected early and treated promptlywith medications that effectively reduce elevated intraocular pressure,loss of visual function or its progressive deterioration can generallybe ameliorated. Drug therapies that have proven to be effective for thereduction of intraocular pressure include both agents that decreaseaqueous humor production and agents that increase the outflow facility.

SUMMARY

This document provides methods and materials related to treatingglaucoma, ocular hypertension, cardiovascular diseases, and renaldiseases. For example, this document provides isolated nucleic acidmolecules encoding a polypeptide having COX-2 activity as well asisolated nucleic acid molecules encoding a polypeptide havingprostaglandin F_(2α) receptor activity. In addition, this documentprovides viral vectors (e.g., lentiviral vectors) containing nucleicacid encoding a polypeptide having COX-2 activity, a polypeptide havingprostaglandin F_(2α) receptor activity, a polypeptide havingprostacyclin IP receptor activity, a polypeptide having prostaglandinsynthase activity, a polypeptide having prostacyclin synthase activity,or combinations thereof Such isolated nucleic acid molecules and viralvectors can be used to reduce intraocular pressure and to treatcardiovascular and renal diseases. For example, viral vectors providedherein can be administered to the eye or eyes of a human patient havingelevated intraocular pressure, thereby reducing the patient's risk ofdeveloping glaucoma. Viral vectors provided herein also can beadministered to the heart of a human patient having cardiovasculardisease, thereby reducing the patient's risk of having a myocardialinfarction. In some cases, viral vectors provided herein can beadministered to the kidneys of a human patient having renal disease,thereby reducing the patient's risk of having renal failure.

This document also provides methods and materials for reducingintraocular pressure. For example, the methods provided herein caninclude administering a viral vector such as a lentiviral vector to oneor both eyes. Such methods can be used to treat existing glaucoma or canbe used to slow or prevent the onset of glaucoma. In some cases, themethods and materials provided herein can be used to reduce a humanpatient's risk of developing glaucoma. In some cases, the methods andmaterials provided herein can be used to increase a mammal's ability torespond to an intraocular pressure reducing treatment such asLatanoprost (xalatan) eye drops.

This document also provides methods and materials for treatingcardiovascular and renal diseases. For example, the methods providedherein can include administering a viral vector systemically,administering a viral vector to the heart (e.g., via a catheter), oradministering a viral vector to one or both kidneys (e.g., via aurethral catheter or during dialysis). Such methods can be used toreduce the severity of a symptom of a cardiovascular or renal disease(e.g., hypertension or renal fibrosis) and can be used to reduce theprogression of a cardiovascular or renal disease (e.g., to reduceprogressive loss of function of the heart or kidneys).

In general, one aspect of this document features a method for treating amammal having glaucoma or elevated intraocular pressure. The methodcomprises, or consists essentially of, administering a nucleic acid toan eye of the mammal under conditions effective to reduce intraocularpressure of the eye, wherein the nucleic acid comprises a nucleic acidsequence encoding a polypeptide having cyclooxygenase-2 activity and anucleic acid sequence encoding a polypeptide having prostaglandin F_(2α)receptor activity. The nucleic acid can be administered to said eyeusing a viral vector (e.g., a lentiviral vector).

In another aspect of this document features a method for treating amammal having glaucoma or elevated intraocular pressure. The methodcomprises, or consists essentially of, administering a viral vector toan eye of the mammal under conditions effective to reduce intraocularpressure of the eye, where the viral vector comprises a nucleic acidencoding a polypeptide having cyclooxygenase-2 activity. The mammal canbe a human. The viral vector can be a lentiviral vector. Theadministering step can comprise contacting the eye with a solutioncontaining the viral vector. The solution can be a saline solution or aphysiologically acceptable buffered solution. The solution can comprisebetween 10³ and 10¹² lentivirus particles per mL (e.g., between 10⁴ and10¹¹ lentivirus particles per mL; between 10⁵ and 10¹⁰ lentivirusparticles per mL; between 10⁶ and 10¹⁰ lentivirus particles per mL; orbetween 10⁶ and 10⁹ lentivirus particles per mL). The nucleic acid canbe a template for an mRNA molecule encoding the polypeptide, where themRNA has an increased stability in cells as compared to the stability ofan mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2.The nucleic acid can comprise a nucleic acid sequence that encodes thesame amino acid sequence as set forth in SEQ ID NO:1 and can comprise acodon sequence different than the codons set forth in SEQ ID NO:2. Thenucleic acid sequence can comprise five or more different codonsequences compared to the codon sequences set forth in SEQ ID NO:2. Thenucleic acid can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1. Theviral vector can comprise a nucleic acid sequence encoding a polypeptidehaving prostaglandin F_(2α) receptor activity. The nucleic acid sequenceencoding the polypeptide having prostaglandin F_(2α) receptor activitycan be a template for an mRNA molecule encoding the polypeptide havingprostaglandin F_(2α) receptor activity, where the mRNA has an increasedstability in cells as compared to the stability of an mRNA moleculetranscribed from the sequence set forth in SEQ ID NO:5. The nucleic acidsequence encoding the polypeptide having prostaglandin F_(2α) receptoractivity can comprise a nucleic acid sequence that encodes the sameamino acid sequence as set forth in SEQ ID NO:4 and can comprise a codonsequence different than the codons set forth in SEQ ID NO:5. The nucleicacid sequence encoding the polypeptide having prostaglandin F_(2α)receptor activity can comprise five or more different codon sequencescompared to the codon sequences set forth in SEQ ID NO:5. The nucleicacid sequence encoding the polypeptide having prostaglandin F_(2α)receptor activity can comprise the sequence set forth in SEQ ID NO:6.The polypeptide having prostaglandin F_(2α) receptor activity cancomprise the sequence set forth in SEQ ID NO:4. The viral vector cancomprise a nucleic acid sequence encoding a polypeptide havingprostaglandin synthase activity. The nucleic acid sequence encoding thepolypeptide having prostaglandin synthase activity can comprise thesequence set forth in SEQ ID NO:8. The polypeptide having prostaglandinsynthase activity can comprise the sequence set forth in SEQ ID NO:7.The viral vector can comprise nucleic acid encoding a polypeptide havingprostaglandin F_(2α) receptor activity and a polypeptide havingprostaglandin synthase activity. The method can be effective to reducethe intraocular pressure by at least 10 percent. The method can beeffective to reduce the intraocular pressure by at least 20 percent. Themethod can be effective to reduce the intraocular pressure by at least30 percent. The viral vector can be a feline immunodeficiency virusvector.

In another aspect, this document features a method for treating a mammalhaving a cardiovascular or renal disease. The method comprises, orconsists essentially of, administering a viral vector to the mammalunder conditions effective to reduce the severity of a symptom of thecardiovascular or renal disease, where the viral vector comprises anucleic acid encoding a polypeptide having cyclooxygenase-2 activity.The mammal can be a human. The viral vector can be a lentiviral vector.The nucleic acid can be a template for an mRNA molecule encoding thepolypeptide, where the mRNA has an increased stability in cells ascompared to the stability of an mRNA molecule transcribed from thesequence set forth in SEQ ID NO:2. The nucleic acid can comprise anucleic acid sequence that encodes the same amino acid sequence as setforth in SEQ ID NO:1 and can comprise a codon sequence different thanthe codons set forth in SEQ ID NO:2. The nucleic acid sequence cancomprise five or more different codon sequences compared to the codonsequences set forth in SEQ ID NO:2. The nucleic acid can comprise thesequence set forth in SEQ ID NO:3. The polypeptide can comprise thesequence set forth in SEQ ID NO:1. The viral vector can comprise anucleic acid sequence encoding a polypeptide having prostacyclin IPreceptor activity. The nucleic acid can comprise a nucleic acid sequencethat encodes the same amino acid sequence as set forth in SEQ ID NO:9and can comprise a codon sequence different than the codons set forth inSEQ ID NO:10. The nucleic acid sequence encoding the polypeptide havingprostacyclin IP receptor activity can comprise the sequence set forth inSEQ ID NO:10. The polypeptide having prostacyclin IP receptor activitycan comprise the sequence set forth in SEQ ID NO:9. The viral vector cancomprise a nucleic acid sequence encoding a polypeptide havingprostacyclin synthase activity. The nucleic acid sequence encoding thepolypeptide having prostacyclin synthase activity can comprise thesequence set forth in SEQ ID NO:12. The polypeptide having prostacyclinsynthase activity can comprise the sequence set forth in SEQ ID NO:11.The viral vector can comprise nucleic acid encoding two or morepolypeptides selected from the group consisting of a polypeptide havingcyclooxygenase-2 activity, a polypeptide having prostacyclin IP receptoractivity, and a polypeptide having prostacyclin synthase activity. Thesymptom can be reduced by 25%. The symptom can be reduced by 50%. Thesymptom can be reduced by 75%. The symptom can be reduced by 100%.

In another aspect, this document features a viral vector comprising anucleic acid encoding a polypeptide having cyclooxygenase-2 activity.The viral vector can be a lentiviral vector. The nucleic acid can be atemplate for an mRNA molecule encoding the polypeptide, where the mRNAhas an increased stability in cells as compared to the stability of anmRNA molecule transcribed from the sequence set forth in SEQ ID NO:2.The nucleic acid can comprise a nucleic acid sequence that encodes thesame amino acid sequence as set forth in SEQ ID NO:1 and can comprise acodon sequence different than the codons set forth in SEQ ID NO:2. Thenucleic acid sequence can comprise five or more different codonsequences compared to the codon sequences set forth in SEQ ID NO:2. Thenucleic acid can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1. Theviral vector can comprise a nucleic acid sequence encoding a polypeptidehaving prostaglandin F_(2α) receptor activity. The nucleic acid sequenceencoding the polypeptide having prostaglandin F_(2α) receptor activitycan be a template for an mRNA molecule encoding the polypeptide havingprostaglandin F_(2α) receptor activity, where the mRNA has an increasedstability in cells as compared to the stability of an mRNA moleculetranscribed from the sequence set forth in SEQ ID NO:5. The nucleic acidsequence encoding the polypeptide having prostaglandin F_(2α) receptoractivity can comprise a nucleic acid sequence that encodes the sameamino acid sequence as set forth in SEQ ID NO:4 and can comprise a codonsequence different than the codons set forth in SEQ ID NO:5. The nucleicacid sequence encoding the polypeptide having prostaglandin F_(2α)receptor activity can comprise five or more different codon sequencescompared to the codon sequences set forth in SEQ ID NO:5. The nucleicacid sequence encoding the polypeptide having prostaglandin F_(2α)receptor activity can comprise the sequence set forth in SEQ ID NO:6.The polypeptide having prostaglandin F_(2α) receptor activity cancomprise the sequence set forth in SEQ ID NO:4. The viral vector cancomprise a nucleic acid sequence encoding a polypeptide havingprostaglandin synthase activity. The nucleic acid sequence encoding thepolypeptide having prostaglandin synthase activity can comprise thesequence set forth in SEQ ID NO:8. The polypeptide having prostaglandinsynthase activity can comprise the sequence set forth in SEQ ID NO:7.The viral vector can comprise nucleic acid encoding a polypeptide havingprostaglandin F_(2α) receptor activity and a polypeptide havingprostaglandin synthase activity. The viral vector can comprise nucleicacid encoding a polypeptide having prostacyclin IP receptor activity.The nucleic acid can comprise a nucleic acid sequence that encodes thesame amino acid sequence as set forth in SEQ ID NO:9 and can comprise acodon sequence different than the codons set forth in SEQ ID NO:10. Thenucleic acid sequence encoding the polypeptide having prostacyclin IPreceptor activity can comprise the sequence set forth in SEQ ID NO:10.The polypeptide having prostacyclin IP receptor activity can comprisethe sequence set forth in SEQ ID NO:9. The viral vector of claim 45,where the viral vector comprises a nucleic acid sequence encoding apolypeptide having prostacyclin synthase activity. The nucleic acidsequence encoding the polypeptide having prostacyclin synthase activitycan comprise the sequence set forth in SEQ ID NO:12. The polypeptidehaving prostacyclin synthase activity can comprise the sequence setforth in SEQ ID NO:11. The viral vector can comprise nucleic acidencoding two or more polypeptides selected from the group consisting ofa polypeptide having cyclooxygenase-2 activity, a polypeptide havingprostacyclin IP receptor activity, and a polypeptide having prostacyclinsynthase activity.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 contains graphs plotting the percent base composition versus baseposition in the human COX-2 mRNA (Upper panel), the coding region of thehuman COX-2 mRNA (middle panel), and the codon-optimized COX-2 cDNA(lower panel).

FIG. 2 contains photomicrographs of cells transfected with transferconstructs containing the wild-type COX-2 cDNA (COX2igWF) or thecodon-optimized COX-2 cDNA (XOGWF) upstream of an IRES operably linkedto a GFP coding sequence. Cells transfected with a transfer constructcontaining a GFP coding sequence operably linked to a CMV promoter(GINWF) served as a positive control, and mock transfected cells servedas a negative control.

FIG. 3 is a Northern blot analyzing expression of COX-2 mRNA in cellstransfected with a transfer construct containing the codon-optimized(XOGWF) or wild-type (COX2igWF) COX-2 cDNA. Cells transfected with atransfer construct containing a GFP coding sequence operably linked to aCMV promoter (GINWF) served as a positive control, and mock transfectedcells served as a negative control.

FIG. 4 is a Western blot analyzing expression of COX-2 polypeptides in293T cells transfected with a transfer construct containing acodon-optimized or wild-type COX-2 cDNA.

FIG. 5 is a schematic diagram of FIV-based lentiviral transferconstructs containing a codon-optimized COX-2 cDNA (pXOGWF), a PGFsynthase cDNA (pPGFSigWF), or a codon-optimized prostaglandin F receptorcDNA (pHAFPRigWF). The prostaglandin F receptor cDNA was HA-tagged toenable detection of prostaglandin F receptor polypeptides on Westernblots

FIG. 6 is a Western blot analyzing expression of prostaglandin Freceptor (FPR), COX-2, and prostaglandin F synthase (PGFS) polypeptidesin cells that were mock transfected or transfected with one or morelentiviral transfer vectors containing a cDNA encoding an FPR, COX-2, orPGFS polypeptide.

FIG. 7 is a graph plotting levels of PGF2alpha in 293T cells transfectedwith a construct containing a COX-2 cDNA, in 293T cells transfected witha construct containing a PGF synthase (PGFS) cDNA, and in 293T cellsco-transfected with a construct containing a COX-2 cDNA and a constructcontaining a PGFS cDNA. FIG. 7 also contains Western blots analyzingexpression of COX-2 and PGFS polypeptides in the transfected 293T cells.

FIG. 8 is a chart indicating the therapeutic regimen applied to eachsubject in the animal study.

FIG. 9 contains a series of graphs plotting intraocular pressure (mm Hg)versus days post injection for the indicated treatment groups.

FIG. 10 is a graph plotting the mean intraocular pressure (TOP)sustained for more than two months in each of the five experimentalgroups described in FIG. 8 and in eyes treated with the control vector.The p-values were determined using a paired, two-tailed distributionT-test.

FIG. 11 is a listing of an amino acid sequence (SEQ ID NO:1) of a humanCOX-2 polypeptide.

FIG. 12 is a listing of a wild-type human nucleic acid sequence (SEQ IDNO:2) encoding the amino acid sequence set forth in SEQ ID NO:1.

FIG. 13 is a listing of a codon optimized nucleic acid sequence (SEQ IDNO:3) encoding the amino acid sequence set forth in SEQ ID NO:1. Thebold, underlined nucleotides represent nucleotides that were changedrelative to the sequence set forth in SEQ ID NO:2.

FIG. 14 is a listing of an amino acid sequence (SEQ ID NO:4) of a humanprostaglandin F_(2α) receptor polypeptide containing an HA tag. Theunderlined amino acid sequence represents the HA tag.

FIG. 15 is a listing of a wild-type human nucleic acid sequence (SEQ IDNO:5) encoding the amino acid sequence set forth in SEQ ID NO:4.

FIG. 16 is a listing of a codon optimized nucleic acid sequence (SEQ IDNO:6) encoding the amino acid sequence set forth in SEQ ID NO:4. Thebold, underlined nucleotides represent nucleotides that were changedrelative to the sequence set forth in SEQ ID NO:5.

FIG. 17 is a listing of an amino acid sequence (SEQ ID NO:7) of a humanprostaglandin F synthase polypeptide.

FIG. 18 is a listing of a nucleic acid sequence (SEQ ID NO:8) encoding ahuman prostaglandin F synthase polypeptide.

DETAILED DESCRIPTION

This document provides methods and materials related to treatingglaucoma, intraocular hypertension, cardiovascular disease, and renaldisease. For example, this document provides isolated nucleic acidmolecules encoding a polypeptide having COX-2 activity as well asisolated nucleic acid molecules encoding a polypeptide havingprostaglandin F_(2α) receptor activity. This document also providesviral vectors (e.g., lentiviral vectors) containing a polypeptide havingCOX-2 activity, a polypeptide having prostaglandin F_(2α) receptoractivity, a polypeptide having prostacyclin IP receptor activity, apolypeptide having prostaglandin synthase activity, a polypeptide havingprostacyclin synthase activity, or combinations thereof

In addition, this document provides methods and materials for reducingintraocular pressure. For example, the methods provided herein caninclude administering a viral vector such as a lentiviral vector to oneor both eyes. Such methods can be used to treat existing glaucoma or canbe used to slow or prevent the onset of glaucoma. In some cases, themethods and materials provided herein can be used to reduce a humanpatient's risk of developing glaucoma.

This document also provides methods and materials for treatingcardiovascular diseases (e.g., pulmonary hypertension) and renaldiseases (e.g., diabetic nephropathy). For example, the methods providedherein can include administering a viral vector systemically,administering a viral vector to the heart, or administering a viralvector to one or both kidneys. Such methods can be used to reduce theseverity of a symptom of a cardiovascular or renal disease (e.g.,hypertension or renal fibrosis) and can be used to reduce theprogression of a cardiovascular or renal disease (e.g., to reduceprogressive loss of function of the heart or kidneys).

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

The term “isolated” as used herein with reference to nucleic acid refersto a naturally-occurring nucleic acid that is not immediately contiguouswith both of the sequences with which it is immediately contiguous (oneon the 5′ end and one on the 3′ end) in the naturally-occurring genomeof the organism from which it is derived. For example, an isolatednucleic acid can be, without limitation, a recombinant DNA molecule ofany length, provided one of the nucleic acid sequences normally foundimmediately flanking that recombinant DNA molecule in anaturally-occurring genome is removed or absent. Thus, an isolatednucleic acid includes, without limitation, a recombinant DNA that existsas a separate molecule (e.g., a cDNA or a genomic DNA fragment producedby PCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleic acidsequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acidexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

An isolated nucleic acid molecule provided herein can contain a nucleicacid sequence encoding a polypeptide having COX-2 activity, apolypeptide having prostaglandin F_(2α) receptor activity, a polypeptidehaving prostacyclin IP receptor activity, a polypeptide havingprostaglandin synthase activity, a polypeptide having prostacyclinsynthase activity, or combinations thereof Non-limiting examples ofnucleic acid sequences encoding a polypeptide having COX-2 activity, apolypeptide having prostaglandin F2α receptor activity, a polypeptidehaving prostacyclin IP receptor activity, a polypeptide havingprostaglandin synthase activity, and a polypeptide having prostacyclinsynthase activity are set forth in SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:10 (GenBank® GI Number GI:39995095), SEQ ID NO:8, and SEQ ID NO:12(GenBank® GI Number GI:75517290), respectively. Non-limiting examples ofamino acid sequences of polypeptides having COX-2 activity,prostaglandin F2α receptor activity, prostacyclin IP receptor activity,prostaglandin synthase activity, and prostacyclin synthase activity areset forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:9 (GenBank® GI NumberGI:4506263), SEQ ID NO:7, and SEQ ID NO:11 (GenBank® GI NumberGI:2493373), respectively.

Isolated nucleic acid molecules provided herein can contain a sequencethat has one or more codons that are different from those found in awild-type sequence. For example, an isolated nucleic acid moleculeprovided herein can contain a nucleic acid sequence that encodes apolypeptide having COX-2 activity that is identical to a human COX-2polypeptide with the nucleic acid sequence having one or more codonsthat are different from wild-type human nucleic acid encoding that humanCOX-2 polypeptide. An example of such a nucleic acid molecule isprovided in FIG. 13.

Any method can be used to obtain an isolated nucleic acid moleculeprovided herein including, without limitation, common molecular cloningand chemical nucleic acid synthesis techniques. For example, PCR can beused to obtain an isolated nucleic acid molecule containing a nucleicacid sequence set forth in FIG. 12. In some cases, the obtained nucleicacid can be mutated to form a codon-optimized sequence such as thesequence set forth in FIG. 13.

Any of the nucleic acid molecules provided herein can be incorporatedinto a viral vector. For example, a viral vector can be designed tocontain an isolated nucleic acid molecule having a nucleic acid sequenceencoding a polypeptide having COX-2 activity, a polypeptide havingprostaglandin F2α receptor activity, a polypeptide having prostacyclinIP receptor activity, a polypeptide having prostaglandin synthaseactivity, a polypeptide having prostacyclin synthase activity, orcombinations thereof Examples of viral vectors that can be used include,without limitation, lentiviral vectors (e.g., feline immunodeficiencyviral vectors), retroviral vectors (e.g., murine retroviral vectors),foamy virus vectors, adenovirus vectors, adeno-associated virus vectors,vaccinia virus vectors, and herpes virus vectors. Viral vectors can bereplication incompetent and can contain few if any viral genes. In somecases, the isolated nucleic acid molecules provided herein can used asnaked DNA or can be incorporated into plasmids, transposons,retroelement-based vectors, or phage integrase containing DNA vectors.

This document provides methods for treating glaucoma or intraocularhypertension. Such methods can include administering an isolated nucleicacid molecule provided herein to a mammal in need of treatment (e.g., ahuman, dog, cat, horse, cow, pig, or monkey). Any method can be used toadminister an isolated nucleic acid molecule provided herein. Forexample, a viral vector provided herein can be administered to a mammalvia oral administration or direct administration to one or both eyes. Insome cases, a viral vector provided herein can be contained within asolution that can be directly applied to an eye. Any method can be usedto administer such a solution to an eye. For example, a solutioncontaining a viral vector provided herein can be administered in amanner similar to the manner used to self administer eye drops.

As described herein, this document provides methods and materials fortreating cardiovascular and renal diseases. Cardiovascular diseasesinclude, without limitation, pulmonary hypertension (e.g., pulmonaryarteriolar hypertension), arterial thrombosis, myocardial ischemia,myocardial infarction, atherosclerosis, restenosis, and reperfusioninjury. Examples of renal diseases include, without limitation, diabeticnephropathy, progressive renal disease, renal fibrosis, renalhypertrophy, and glomerulosclerosis. As described herein, a mammalhaving a cardiovascular or renal disease can be treated using anisolated nucleic acid molecule provided herein (e.g., an isolatednucleic acid molecule encoding a polypeptide having cyclooxygenase-2activity, a polypeptide having prostacyclin IP receptor activity, apolypeptide having prostacyclin synthase activity, or any combinationthereof). A viral vector containing an isolated nucleic acid moleculeprovided herein can be used to administer the nucleic acid to a mammalin need of treatment. Viral vectors can be prepared using standardmaterials (e.g., packaging cell lines and vectors) and methods known tothose of ordinary skill in the art.

Viral vectors containing one or more nucleic acid molecules providedherein (e.g., one or more of a nucleic acid encoding a polypeptidehaving cyclooxygenase-2 activity, a nucleic acid encoding a polypeptidehaving prostacyclin IP receptor activity, and a nucleic acid encoding apolypeptide having prostacyclin synthase activity) can be administeredto a mammal having a cardiovascular or renal disease via numerousroutes. For example, a viral vector can be administered systemically(e.g., via intravenous injection). In some cases, a viral vector can beadministered directly to the heart. Direct administration of a viralvector to the heart can be achieved using a catheter or a stent, forexample, and can performed during a therapeutic manipulation such asarterial bypass surgery. In some cases, a viral vector can beadministered directly to one or both kidneys. Various methods can beused to deliver a viral vector to a kidney. For example, a urethralcatheter can be used to deliver a viral vector to a kidney. In somecases, a viral vector can be delivered to a kidney during dialysis or bydirect injection into the kidney (e.g., CT-guided direct needleinjection into the kidney). In some cases, a viral vector can betargeted to the heart or kidneys using liposomes or by expressing apolypeptide on the surface of the viral particle that interacts withanother polypeptide that is expressed predominantly or selectively onthe surface of heart or kidney cells. In some cases, one or more nucleicacid molecules provided herein can be administered to a mammal havingcardiovascular or renal disease by direct injection of the naked nucleicacid molecules into the heart or kidney, or by direct administration ofliposomes containing the nucleic acid molecules. As with viral vectors,liposomes also can be targeted to heart or kidney tissue. Viral vectors,naked nucleic acids, and liposomes can be administered to a mammal in abiologically compatible solution or a pharmaceutically acceptabledelivery vehicle. Suitable pharmaceutical formulations depend in part onthe use and route of delivery. For example, a suitable formulation fordirect injection is isotonic and has a neutral pH.

After identifying a mammal as having glaucoma, intraocular hypertension,a cardiovascular disease, or a renal disease, the mammal can beadministered a viral vector containing a nucleic acid disclosed herein.A viral vector can be administered to a mammal in any amount, at anyfrequency, and for any duration effective to achieve a desired outcome(e.g., to reduce the severity of a symptom of glaucoma, cardiovasculardisease, or renal disease). In some cases, a viral vector can beadministered to a mammal having glaucoma, intraocular hypertension, acardiovascular disease, or a renal disease to reduce the severity of asymptom or to reduce the progression rate of the condition by 5, 10, 25,50, 75, 100, or more percent. For example, the severity of a symptom canbe reduced in a mammal such that the symptom is no longer detected bythe mammal In some cases, the progression of a condition can be reducedsuch that no additional progression is detected. Any method can be usedto determine whether or not the severity of a symptom or the progressionrate of a condition is reduced. For example, a mammal having glaucomacan be tested for intraocular pressure before and after treatment todetermine whether the pressure is reduced. In some cases, a mammal canbe observed or tested for the severity of a symptom of cardiovasculardisease (e.g., high blood pressure, blood clots in arteries and veins,pain isolated to one leg (usually the calf or medial thigh), swelling inthe extremity, or varicose veins) before and after treatment todetermine whether or not the severity of a symptom is reduced. In somecases, renal biopsy tissue taken from a mammal before and aftertreatment can be analyzed (e.g., for fibrosis) to determine whether theseverity of a symptom is reduced. To determine whether or notprogression of a condition (e.g., glaucoma, intraocular hypertension,cardiovascular disease, or renal disease) is reduced, a physicalexamination can be performed at different time points to determine thestage or severity of the condition. The stage or severity of thecondition observed at different time points can be compared to assessthe progression rate. After treatment as described herein, theprogression rate can be determined again over another time interval todetermine whether or not the progression rate has decreased. Forexample, renal function can be assessed at various time points todetermine whether the function is improving, worsening, or staying thesame.

An effective amount of a viral vector can be any amount that reduces theseverity of a symptom or the progression of a condition (e.g., glaucoma,intraocular hypertension, cardiovascular disease, or renal disease)without producing significant toxicity to the mammal If a particularmammal fails to respond to a particular amount, then the amount of theviral vector can be increased by, for example, two fold. After receivingthis higher concentration, the mammal can be monitored for bothresponsiveness to the treatment and toxicity symptoms, and adjustmentsmade accordingly. The effective amount can remain constant or can beadjusted as a sliding scale or variable dose depending on the mammal'sresponse to treatment. Various factors can influence the actualeffective amount used for a particular application. For example, thefrequency of administration, duration of treatment, use of multipletreatment agents, route of administration, immunocompetency of themammal, and severity of the condition may require an increase ordecrease in the actual effective amount administered.

The frequency of administration can be any frequency that reduces theseverity of a symptom or progression rate of a condition withoutproducing significant toxicity to the mammal For example, the frequencyof administration can be from about once in a lifetime to about once amonth. The frequency of administration can remain constant or can bevariable during the duration of treatment. A course of treatment with aviral vector can include rest periods. For example, a viral vector canbe administered over a six month period followed by a three month restperiod, and such a regimen can be repeated multiple times. As with theeffective amount, various factors can influence the actual frequency ofadministration used for a particular application. For example, theeffective amount, duration of treatment, use of multiple treatmentagents, route of administration, immunocompetency of the mammal, andseverity of the condition may require an increase or decrease inadministration frequency.

An effective duration for administering a viral vector provided hereincan be any duration that reduces the severity of a symptom or theprogression rate of glaucoma, intraocular hypertension, cardiovasculardisease, or renal disease without producing significant toxicity to themammal Thus, the effective duration can vary from several days toseveral weeks, months, or years. In general, the effective duration forthe treatment of glaucoma, intraocular hypertension, cardiovasculardisease, or renal disease can range in duration from several months toseveral years. In some cases, an effective duration can be for as longas an individual mammal is alive. Multiple factors can influence theactual effective duration used for a particular treatment. For example,an effective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, immunocompetency of the mammal, and severity of thecondition.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Modulation of Prostaglandin Pathways ReducesIntraocular Pressure

Experiments were conducted to determine whether expression ofpolypeptides involved in prostaglandin biosynthetic and responsepathways can be manipulated to provide a sustained improvement inintraocular pressure involved in diseases such as glaucoma.

Methods and Materials Cloning FIV-Based Transfer Construct Plasmids

pCOX2igWF: A plasmid having the normal human COX-2 cDNA (obtained fromS. Prescott, Huntsman Cancer Institute) was NotI-XbaI-digested toisolate the COX-2 cDNA and blunted with T4 polymerase. pGiNWF (Loewen etal., Investig. Ophthalmol. Vis. Sci., 43:3686-3690 (2002)) was digestedwith Agel and EcoRI, and blunted with T4 polymerase to isolate thebackbone sequence that was then ligated with the COX-2 cDNA insert.Next, an IRES (internal ribosome entry site)-GFP cassette was blunt-endligated into the EcoRI site just downstream of COX-2. This insertionresulted in a bicistronic FIV-based transfer construct with COX-2expression driven by the CMV promoter and GFP expression being driven bythe IRES just downstream of the COX-2 cDNA cassette. This plasmid,COX2igWF, was the basis for the cloning of the following transferconstructs.

pXOGWF: A codon-optimized human COX-2 cDNA was designed and synthesizedwith the assistance of GenScript Corporation custom services (ScotchPlains, NJ). Codon usage was optimized for usage in mammalian cells. G-Ccontent was optimized, and other factors such as secondary structure andrepetitive codons were taken into consideration to achieve codonoptimization. The codon-optimized COX-2 cDNA was designed to includeflanking restriction sites to enable downstream recombinant cloningstrategies. BamHI sites flank the codon-optimized COX-2 gene, and thesesites were used to insert the optimized cDNA into the BamHI-digestedbackbone of COX2igWF. This essentially replaced the wild-type COX-2 cDNAcassette with the codon-optimized COX-2 cDNA.

pGFSigWF: The PGFS cDNA from hPGFS-cDNA pUC8 (obtained from KikukoWatanabe, University of East Asia, Japan) was removed by EcoRI and Salldigestion, and blunted with T4 DNA polymerase. This cDNA insert wasblunt ligated into the BamHI-digested backbone of COX2igWF.

pHAFPRigWF: An HA-tagged prostaglandin F receptor (HAFPR) cDNA plasmid(obtained from G. FitzGerald, University of Pennsylvania, USA), HA-FP,contains the HAFPR cDNA flanked by an upstream KpnI and a downstreamNcoI site. The HAFPR cassette was removed by KpnI-NcoI digestion,blunted with T4 DNA polymerase and ligated with T4 DNA ligase into theBamHI-digested backbone of COX2igWF.

pcoFPRigWF: A codon-optimized human HA-tagged FPR cDNA was designed andsynthesized with the assistance of GenScript Corporation custom services(Scotch Plains, N.J.). Codon usage was optimized for usage in mammaliancells. G-C content was optimized, and other factors such as secondarystructure and repetitive codons were taken into consideration to achievecodon optimization. The codon-optimized HAFPR was designed to includeflanking restriction sites for accessible cloning strategies. BamHIsites flank the codon-optimized HAFPR gene and were used to digest andligate into the BamHI-digested backbone of COX2igWF.

PGF2Alpha Assay

293T cells in 6 well plates were transfected with 2 μg XOGWF or PGFSigWFusing the calcium phosphate transfection method as described elsewhere(Loewen et al., Methods Mol. Biol., 229:251-271 (2003)). Media waschanged 12 to 16 hours later and collected 24 hours thereafter. Mediawas filtered (0.2 μm) to remove cells and used in the Prostaglandin F2 αenzyme immunoassay kit (Cayman Chemical, cat. no. 516011) as recommendedby the manufacturer's protocol manual.

Northern Blot

293T cells were transfected with equivalent quantities of COX2igWF,XOWGF, or GINWF. Cells were treated with 1 mL Trizol 36 hourspost-transfection and stored at −80° C. until RNA purification.Trizol-treated lysates were treated with chloroform, followed byisopropanol, and spun to isolate nucleic acid. Nucleic acid was treatedwith DNase, followed by RNA extraction with equal volume ofphenol:chloroform:isoamyl alcohol (125:24:1). RNA was precipitated with1/10 volume 3M sodium acetate and 2.5 volumes 100% ethanol.

Isolated total cellular RNA was separated by gel electrophoresis (1.2%agarose gel, 3.75% formaldehyde, 1× MOPS). After gel electrophoresis,RNA was transferred onto a nylon transfer membrane (Nytran SuperchargeMembrane, Schleicher & Schuell, cat. no. 10416284).

A beta-actin anti-sense oligo probe was 5′-end labeled using T4 PNK(Promega) and [γ-³²P]ATP incubated at 37° C. for 30 minutes followed byheat inactivation at 70° C. for 10 minutes. Labeled primer probe waspurified using a quick spin column and hybridized with RNA on nylonmembrane overnight at 42° C. The membrane was washed and then exposed toMR X-ray film (Kodak) at -80° C. for five days and developed.

Since all transfer constructs contain the GFP gene cassette to beincluded in the message, mRNA expression levels for each message wereassessed by probing for GFP sequence common to all messages in thisexperiment.

25 ng of GFP anti-sense oligo probe was randomly labeled using dNTPstock solution, 50 μCi a-dCTP and Klenow. The random labeling reactionoccurred at 37° C. for 1 hour and was heat-inactivated at 65° C. for 10minutes. Random labeled probe was purified using a quick spin columnfollowed by hybridization with the RNA-containing nylon membrane for 5hours to 3 days at −80° C. The membrane was exposed to MR X-ray film(Kodak) and developed.

Western Blot

For Western blotting, cells were lysed in Tris-buffered salinecontaining 1% Triton X-100 and 1% NP-40, plus a protease inhibitorcocktail (Complete-mini; Boehringer). Lysates were centrifuged to removechromatin. Proteins were resolved in sodium dodecyl sulfate-10%polyacrylamide gels and transferred to Immobilon P membranes(Millipore). Blocked membranes were incubated overnight at 4° C. or for2 hours at room temperature with mouse anti-COX-2 MAb (Cayman Chemical,cat. no. 160112), rat anti-HA MAb (Roche), or rabbit anti-hPGFS Ab(obtained from Kikuko Watanabe), diluted in Tris-buffered saline-5%nonfat milk plus 0.05% Tween 20. After washing, membranes were incubatedwith the appropriate horseradish peroxidase-tagged secondary antibody.Bound antibodies were detected by ECL (Amersham Pharmacia Biotech).

Vector Production and Titration

Transfections were performed using the calcium phosphate transienttransfection method in ten-chamber cell factories (CF10) as describedelsewhere (Loewen et al., Methods Mol. Biol., 229:251-271 (2003)).Medium was changed 12 to 16 hours later, and supernatants were collected48 hours thereafter, filtered through a 0.2-μm-pore-size filter, andconcentrated by two rounds of ultracentrifugation. The first spin wasperformed in a series of 250 mL polyallomer Oakridge ultracentrifugebottles (Sorvall, cat. no. 54477) at 19,000 rpm in a SureSpin 630 rotor(Sorvall, cat. no. 79367) in a Sorvall Discovery 100SE ultracentrifuge(67,000 g_(max)) for 6 hours at 4° C. Supernatant was removed, andvector was resupended in 30 mL PBS and centrifuged over a sucrosecushion in a swinging bucket SW41TI rotor at 24,000 rpm for 2 hours at4° C., and aliquoted and frozen at −80° C.

CrFK cells were transduced with serial dilutions of each vectorpreparation. 48 hours after transduction, cells were harvested, andtiters of each vector preparation were determined by flow cytometry forGFP expression. All preparations were tested for reverse transcriptase(RT) activity as described elsewhere (Saenz et al., J. Virol.,79(24):15175-88 (2005)).

Vector Administration to Cat Anterior Chamber

Experiments were conducted in pathogen-free domestic cats (Harlan,Indianapolis, IN). Prior to vector administration, cats wereanesthetized with 10 mg/kg intramuscular tiletamine HCl/zolazepam HCl(Telazol; Fort Dodge Laboratories Inc., Fort Dodge, Iowa) injection.Anterior chambers of feline eyes were transcorneally injected with abolus of 200 μL PBS containing 10⁷ TU of vectors GINWF, XOGWF, PGFSigWF,or HAFPRigWF. Animals receiving two or more different vectors received atotal of 2×10⁷ TU and 3×10⁷ TU vector, respectively.

Intraocular Pressure Measurements, Slit Lamp Examinations, & GonioscopicObservation

Prior to examinations, cats were anesthetized with 10 mg/kgintramuscular tiletamine HCl/zolazepam HCl (Telazol; Fort DodgeLaboratories Inc., Fort Dodge, Iowa) injection. Weekly examinationsconsisted of slit lamp (Haag-Streit, Mason, Ohio) observation anddetermination of intraocular pressure using a handheld pneumatonometer(Model 30 Classic; Medtronic, Fridley, Minn.).

Fluorescence of transduced TM was observed with a standard gonioscope(Posner; Ocular Instruments, Bellevue, Wash.) and a microscope (EclipseE400; Nikon) equipped with a GFP-optimized filter (EF-4 B-2E/C FITC,cat. no. 96107; Nikon).

Results

Codon Optimization of Prostaglandin Pathway mRNAs

Initial studies revealed that human COX-2 and PGF receptor polypeptideswere difficult to express using standard methods. By performing insilico analyses, it was discovered that the coding regions of both theCOX-2 and PGF receptor mRNAs were aberrantly AU-rich, with a markedlysuboptimal codon bias. The skewed codon use of the human COX-2 codingregion is very similar in composition to that of lentiviral structuralgenes. This composition makes lentiviral mRNAs labile, a problem thatthe viruses overcome with specialized viral polypeptides that stabilizeRNA at particular stages of the life cycle. The composition of theprostaglandin pathway mRNAs presumably fosters rapid endogenousturnover. Human codon-optimized versions of the human COX-2 cDNA(FIG. 1) and the PGF receptor cDNA were synthesized. The codon-optimizedCOX-2 cDNA contains a GC-rich sequence that encodes an amino acidsequence identical to the wild-type COX-2 sequence.

Transfer constructs were generated that contained the wild-type or thecodon-optimized COX-2 cDNA upstream of an IRES operably linked to a GFPcoding sequence. Cells were transfected with the constructs, and GFPexpression levels were observed. A significantly higher level of GFPexpression was observed in cells transfected with the constructcontaining the codon-optimized COX-2 cDNA (XOGWF) as compared to cellstransfected with the construct containing the wild-type COX-2 cDNA(COX2igWF; FIG. 2).

Cells transfected with the transfer constructs containing the wild-typeor codon-optimized COX-2 cDNA were also analyzed for mRNA levels byNorthern blotting. The blots were analyzed with a GFP proberandom-labeled using ³²P-dCTP. The blots were also analyzed with aβ-actin probe 5′-labeled using ³²P-dATP to control for equal loading.The level of mRNA was much higher in cells transfected with theconstruct containing the codon-optimized COX-2 cDNA than in cellstransfected with the construct containing the wild-type COX-2 cDNA (FIG.3).

Recombinant DNA constructs containing the codon-optimized COX-2 cDNA orthe wild-type COX-2 cDNA were also used to transfect 293T cells, andlysates from the transfected cells were analyzed for COX-2 expression byWestern blotting. Expression of COX-2 polypeptides was higher in cellstransfected with the construct containing the codon-optimized COX-2 cDNAthan in cells transfected with the construct containing the wild-typeCOX-2 cDNA (FIG. 4).

These results indicate that codon optimization of the COX-2 codingregion increases the stability of the transcribed RNA, resulting inincreased expression at the polypeptide level. The wild-type COX-2coding region, not just the 3′ untranslated region as was previouslyrecognized, prevents significant polypeptide expression. In contrast,PGF synthase does not have an aberrant RNA base composition and does notrequire codon optimization.

Effect of the Expression of Prostaglandin Pathway Polypeptides on IOP invivo

Lentiviral transfer constructs based on the feline immunodeficiencyvirus (FIV) vector system (Poeschla et al., Nat. Med., 4(3):354-7(1998)) were generated which contained a human codon-optimized COX-2cDNA, a human PGF synthase cDNA, or a human codon-optimized PGF receptorcDNA (FIG. 5). Levels of COX-2, PGF synthase, and PGF receptorpolypeptides in cells transfected with one or more of the transferconstructs were analyzed by Western blotting, and expression of each ofthe polypeptides was detected (FIG. 6).

Production of PGF2alpha was measured in 293T cells transfected with aconstruct containing a COX-2 or a PGF synthase (PGFS) cDNA, and in 293Tcells co-transfected with a construct containing a COX-2 cDNA and aconstruct containing a PGF synthase cDNA. Production of PGF2alpha wasobserved in the presence of COX-2 polypeptides and correlated stronglywith the expression level of COX-2 polypeptides (FIG. 7). Co-expressionof COX-2 and PGFS resulted in an even greater level of PGF2alphaproduction than expression of COX-2 alone (FIG. 7). Synthesis ofPGF2alpha was increased up to 0.9×10⁴-fold in the transfected cellsrelative to synthesis of PGF2alpha in control cells. Expression of PGFSalone did not increase PGF2alpha levels, indicating that COX-2 is arate-limiting polypeptide in the prostaglandin synthesis pathway.

The effect of expression of prostaglandin pathway polypeptides onintraocular pressure (TOP) was investigated in a large animal modeldeveloped for glaucoma studies and described elsewhere (Loewen et al.,Invest. Ophthalmol. Vis. Sci., 43(12):3686-90 (2002)). Fifteen domesticcats were divided into five groups, with three cats in each group. Theanterior chamber of the right eye of each cat was injected with one ormore lentiviral vectors containing a COX-2, PGFS, or prostaglandin Freceptor (FPR) cDNA. The anterior chamber of the left eye of each catwas injected with 10⁷-10⁸ TU of a control eGFP vector (FIG. 8). Theanimals were monitored serially for intraocular pressure (TOP) andclinical effects.

The lentiviral vectors were well-tolerated in the animals and producedmarked, sustained (two months at present, with observation of allanimals ongoing), and highly significant IOP decreases (the mean overthe entire two months was 4 2 mm Hg, p<0.002) compared to the IOP levelsin eyes treated with the control vector. A combination of vectorscontaining COX-2 and PGF receptor cDNAs produced the largest IOPdecrease (mean=5.6 mm Hg, 38% reduction, p<5×10⁻¹⁴; FIGS. 9 and 10).

These results indicate that major prostaglandin biosynthetic andresponse pathways can be manipulated. Codon optimization of the COX-2coding region profoundly augments mRNA stability. Sustained,substantial, highly statistically significant decreases in IOP wereachieved in a large animal model.

Othere Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method for treating a mammal having glaucoma orelevated intraocular pressure, said method comprising administeringnucleic acid encoding a polypeptide having cyclooxygenase-2 activity toan eye of said mammal under conditions effective to reduce intraocularpressure of said eye by at least 10 percent.
 3. The method of claim 2,wherein said mammal is a human.
 4. The method of claim 2, wherein saidadministering step comprises contacting said eye with a solutioncontaining said nucleic acid encoding said polypeptide havingcyclooxygenase-2 activity.
 5. The method of claim 2, wherein said methodis effective to reduce said intraocular pressure by at least 20 percent.6. The method of claim 2, wherein said method is effective to reducesaid intraocular pressure by at least 30 percent.
 7. The method of claim2, wherein said nucleic acid encoding said polypeptide havingcyclooxygenase-2 activity is administered using a viral vector.
 8. Themethod of claim 7, wherein said viral vector is a lentiviral vector. 9.The method of claim 2, wherein said polypeptide having cyclooxygenase-2activity is a human cyclooxygenase-2 polypeptide, and wherein saidnucleic acid encoding said polypeptide having cyclooxygenase-2 activityis codon-optimized for use in mammalian cells.